Acquired Immune Deficiency Syndrome, generally known by its acronym AIDS, is probably the most serious health threat confronting society. The disease runs a painful and debilitating course and usually results in the death of its victim. In fact, from diagnosis onward, the average life span of an AIDS victim is less than two years,
To date, about 40,000 AIDS cases have been reported in the United States. Approximately two-thirds of individuals have died from the disease.
AIDS is caused by a virus which has at various times been called human T-cell lymphotropic virus type III (HTLV III), or lymphoadenopathy-associated virus (LAV). The virus is currently known as human immunodeficiency virus I (HIV-1). It is estimated by the Center for Disease Control, U.S. Public Health Services, and the National Academy of Sciences that in the United States alone, about 1.5 million people will have been infected by 1991. The results from many long-term epidemiological studies indicate that twenty to sixty percent of the infected group will develop AIDS within the next five to seven years. For example, the Center for Disease Control has estimated that there will about 300,000 AIDS cases by the 1991.
HIV-1 also causes a somewhat less serious immunodeficiency syndrome clinically defined as AIDS related complex (ARC). ARC will often precede the onset of AIDS. There are currently many more ARC cases than there are AIDS cases. As the number of cases continues to increase, ARC will, in and of itself, become an extremely costly and serious health problem.
AIDS results because infection with HIV-1 damages and eventually destroys the victim's immune system. The immune system is reduced to the point where the victim can no longer ward off secondary opportunistic infections. It is often the secondary infections which debilitate the victim and cause death.
In addition to their susceptibility to secondary infections, AIDS victims frequently develop otherwise rare conditions. A large number develop a rare form of skin cancer known as Kaposi's sarcoma. It is believed that this condition also results from the immunodeficiency brought on by the virus.
HIV-1 damages the immune system by infecting and depleting T helper/inducer lymphocytes (hereinafter referred to "T cells"). T cells are essential because they control the production of antibodies by the B cells, the maturation of cytotoxic T lymphocytes (killer T cells), the maturation and activity of macrophages and natural killer cells, and, directly and indirectly, numerous other regulator and effector functions of the immune system.
Infection of a T cell occurs through interaction between an epitope borne by HIV-1 and a receptor site which is located on the T cell surface. This receptor site on the T cell is protein molecule known as the CD4 antigen. The epitope on HIV-1 is borne by the envelope glycoprotein gp 120 (molecular weight 120,000 daltons). The glycoprotein gp 120 is produced when a precursor glycoprotein gp 160, made in the T cell, is cleaved apart into gp 41 (molecular weight 41,000 daltons) and gp 120. Gp 41 bears the epitope which induces the dominant antibody response in most infected individuals, whereas the epitope borne by gp 120 binds to the CD4 antigen and thereby allows the virus to enter the cell.
HIV-1 is a retrovirus. After the virus has entered the cell, a viral enzyme called reverse transcriptase transcribes the viral genomic RNA into DNA in the host cell nucleus. The newly synthesized DNA acts as a template and causes the infected T cell to begin to transcribe the new DNA to make copies of messenger RNA and genomic RNA. The viral genomic RNA's are packed with core proteins, reverse transcriptase, and certain other proteins. They are then enveloped by parts of the cellular membrane and budded off from the cell into the bloodstream as newly synthesized virions. These new virions can enter and infect other T cells.
There are two known mechanisms by which HIV-1 is transmitted to T cells in the body of infected individuals. The first occurs when the free virus binds to the CD4 antigen on the T cells. The second mechanism is through direct, cell-to-cell transmission of the virus.
Direct, cell-to-cell transmission occurs when an infected cell, which expresses the viral gp 120 on its surface, binds with the CD4 antigen of an uninfected cell. As a result the two cells fuse and virions can pass to the uninfected cell.
Direct, cell-to-cell contact and the resulting fusion are a significant source of cellular infection, and may be a major mechanism of T cell destruction in HIV-1 infected individuals. Infected and uninfected cells often fuse in large groups, thereby forming multi-nucleated aggregates known as syncytia. The cell fusion causes the death of cells in the syncytia. See Lifson et al. "Induction of CD4-Dependent Cell Fusion by the HTL-III/LAV Envelope Glycoprotein", Nature 323:725-27 (1986).
The majority of cell death is believed to take place in syncytia. This theory follows because it seems unlikely that significant infection can occur from other sources, such as free virus in the bloodstream. Concentrations of free virus in the bloodstream of infected individuals are typically very low. It also seems unlikely that significant cell infection can occur from discrete fusion of individual infected and uninfected cells. In one study it was found that the proportion of infected T cells in infected individuals is usually only one out of every 10,000 to 100,000 white blood cells. Nevertheless it was reported that the number of CD4 positive cells (i.e., T cells) gradually decreased.
Patients who are infected by HIV-1 do not generate sufficient amounts of neutralizing antibodies. They typically have very low titers of neutralizing antibodies in their serum. Thus, monoclonal antibodies which neutralize HIV-1 would be particularly useful for treatment.
Monoclonal antibodies are produced by hybridoma cells. Hybridomas are cells which have all been cloned from a single fused cell. All the clones are identical to the parent. Accordingly, all the hybridomas of the same clone produce identical antibodies which bind to the same epitope.
A method of making monoclonal antibodies was first described by Koehler and Milstein. See Milstein et al., Nature 256:495-97 (1975); Koehler et al., Eur J. Immunol., 6:511-19 (1976). A host animal, usually a mouse, is immunized with an antigen and then sacrificed. Lymphocytes containing B-cells are then removed, usually from the spleen or other lymphoid tissues. The removed lymphocytes are fused with myeloma cells to form hybridomas. The hybridomas which produce antibody against the designated epitopes of the immunizing antigen are cloned and screened. These hybridomas are then used to manufacture the desired monoclonal antibodies.
A monoclonal antibody which inhibits infectivity and syncytium formation would have many advantages over other neutralizing agents. Large quantities of the monoclonal antibody could be produced. The hybridomas are immortal due to the fusion with myeloma cells, and can be reproduced almost endlessly.
Another advantage of monoclonal methodology is that monoclonal antibodies of high specificity and high affinity can be screened from a large number of antibodies of diverse reactivities and affinities. If one can obtain antibody of high specificity and high affinity, this may allow therapeutic use of the antibody in minimal quantities which are just sufficient enough to bind the appropriate epitopes to neutralize the virus and to prevent syncytia formation.
The high specificity of monoclonal antibodies is to be contrasted with that of other neutralizing agents. In one study, antisera was collected from goats which had been immunized with various proteins from the envelope of HIV-1, including gp 120. The antisera effectively blocked infection of HIV-1 only at low dilutions. See S. D. Putney et al., "HTLV-III/LAV-Neutralizing Antibodies to an E. coli-Produced Fragment of the Virus Envelope", Science 234:1392-95 (1986). Similarly, antisera from rabbits and guinea pigs which were immunized with recombinant gp 120 was effective for HIV-1 neutralization only at low dilutions. See L. A. Lasky et al., "Neutralization of the AIDS Retrovirus by Antibodies to a Recombinant Envelope Glycoprotein", Science 233:209-212 (1986). The polyclonal antibodies used in these studies are non-specific and therefore had to be used in relatively large quantities.
The above results suggest that entire gp120 and long recombinant peptides can not induce high titer neutralization antibodies probably because the "neutralization eptiopes" are not immunogenic. Moreover, the antibodies are found to be typespecific and not group-specific, i.e. they react with only the immunizing HIV-1 strain and not with other strains that are genetically significantly different.
In order for a monoclonal antibody to be used for therapeutical and prophylactic purposes in AIDS, it must exhibit protective activity against diverse HIV-1 strains and a large numver or a significant proportion of field HIV-1 isolates.
In summary, a monoclonal antibody of potential therapeutical value to treat patients with AIDS or ARC and of protective value in preventing AIDS in asymptomatic healthy HIV-1 infected individuals or in preventing HIV-1 infection in individuals of high-risk groups is one that inhibits infection of susceptible cells by broad strains of HIV-1 either via attack by free virions or by direct cell-to-cell transmission (syncytium formation).